Fluorescence in the emission of light (a photon) by a substance that has absorbed light or other electromagnetic radiation. Absorbance of energy excites an orbital electron of a molecule to higher electronic states and relaxation to ground state emits a photon. It is a form of luminescence. Usually, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation.
Chemiluminescence is the emission of light (luminescence), as the result of a chemical reaction.
Fluorophores absorb light energy at one wavelength and, in response, re-emit light energy at another, longer wavelength. Each fluorophore has a distinctive range of wavelengths at which it absorbs light and another distinct range of wavelengths at which it emits light. This property enables their use for specific detection of biological products by analytical instruments and techniques.
Different sample measuring methods based on photoluminescence are known from prior art, wherein emission of light from the sample is obtained with an excitation of light into the sample. When the light emitted by the sample is measured, different properties of the sample can be determined. Known measurement methods include AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) photochemical measurement technology and LOCI (Luminescent Oxygen Channeling Immunoassay) described in U.S. Pat. No. 6,406,913 to Ullman et al., where an emission of light is produced both by excitation of light and by a chemical reaction within the sample.
The light source generally used in AlphaScreen measurement technology method and devices is a laser due to the uniformity of the wavelength of the light produced and the obtained high power density of the light, which high excitation energy effectively causes formation of modified oxygen molecules from beads coated with a luminescent agent. The modified oxygen molecules ultimately initiate a cascade of chemical reactions leading to the generation of the detected chemiluminescent signal.
The AlphaScreen measurement method is a bead-based proximity assay, where Donor beads and Acceptor beads connect through various biological analyte material, which may specifically react with each other and the beads. When the connected Donor beads are excited with a red laser light at 670-690 nm wavelength, they convert ambient oxygen to an excited singlet state. The released singlets of oxygen diffuses to the surrounding sample well matrix and if the Donor and Acceptor beads are connected via the specific biological reaction to be examined, the Acceptor beads are near enough to be excited by the singlets and a light emission is produced between 520-620 nm wavelength, which is a blue-green, green or yellow light and is proportional to the level of interaction. By measuring the emission light, it is possible to establish how effectively the chemical reaction has taken place and what are the properties of the sample being assayed.
The difference in wavelengths between the excitation light and emission light from the sample allows separation and detection of the emitted light from the excitation light.
In the AlphaScreen method, the wavelengths of excitation light and emission light have a reverse relationship compared to fluorescence in general, i.e., excitation light wavelength is longer than the emission light wavelength.
In fluorescence measurement generally, an excitation light impulse is sent to the sample, and measurement of the emission light is carried out effectively without induced delays. In Time-Resolved Fluorescence spectroscopy (TRF), the sample is monitored as a function of time after excitation light pulse or pulses. TRF is utilized to reduce background fluorescence and, for example, in kinetic studies.
Laser is potentially a precise and powerful light source that usually generates a collimated light beam which reduces the need to use separate collimating means in the analytical instrument but the use of a laser as an excitation light source also causes some problems. The high electrical power needed to produce the laser light also generates heat but, since the laser diode's resonators are highly sensitive to self-generated heat and temperature variations, the laser diode requires often active cooling by a separate device, such as a Peltier cooler, which results in added complexity and size of the analytical device. Further, laser diodes are also very expensive, which raises the price of the analytical devices utilizing lasers. In practice, it has been observed that only a few measurements, typically only one, can be performed when the sample is excited with a laser light source in, e.g., AlphaScreen applications because strongly reduced or no emission signal is gained in the subsequent measurements.
Fluorescence reading instruments can be stabilized by using suitable stable fluorescence reference material which converts shorter wavelength excitation radiation to longer wavelength emission radiation. These kinds of materials are widely available and are used, for example, in Thermo Scientific's Fluoroskan Ascent microplate fluorometers.
In Alphascreen measurement, the excitation wavelength (685 nm) is longer than the emission wavelength (520 nm-620 nm). There are no suitable stabile materials available which would convert the excitation wavelength of 685 nm to shorter wavelengths. This is problematic, since the shifting towards shorter wavelengths is in fact one of the benefits of Alphascreen measurement, since no optical background signal is present in the measurement. Also, the used reference materials cannot be used as a reference fluorescence material in TRF measurements, including Alphascreen chemistry, due to lack of TRF behavior.